Manganese and the regulation of its acquisition and subsequent metabolism are important for normal cell function, and contribute to the success of bacteria in adapting to the diverse environments in which they occupy. Manganese can be a limiting nutrient, and thus manganese-dependent processes must be coordinated with availability of the metal. We are interested in understanding how Bradyrhizobium japonicum senses and acquires manganese, maintains homeostasis, and coordinates manganese-dependent activities with cellular levels. Preliminary evidence lead us to test the hypothesis that B. japonicum adapts to cellular manganese levels by novel mechanisms that differ substantially from that found in E. coli and other model organisms. B. japonicum belongs to the alpha-Proteobacteria, a diverse taxonomic group that includes numerous members that form close or intracellular associations with eukaryotic hosts in a symbiotic or pathogenic context. B. japonicum serves as a model organism to study related pathogens that are refractory to experimental analysis. Three specific aims are proposed. (1) Elucidate the mechanisms that maintain manganese homeostasis. We identified a high affinity outer membrane channel necessary for Mn2+ uptake. Outer membrane selectivity for a divalent ion is unprecedented and reveals a bacterial strategy for nutrient acquisition not previously appreciated. We will elucidate the role of this channel in manganese homeostasis. Moreover, B. japonicum tolerates an unusually high external manganese concentration that is toxic to other bacteria. We will determine the basis of this tolerance towards the goal of understanding how this organism maintains homeostasis. (2) Elucidate manganese-responsive transcriptional control via the B. japonicum Mur protein. B. japonicum has adapted a global bacterial iron-responsive transcriptional repressor to function as a Mn2+ responsive regulator. We uncovered novel aspects of the Mur protein. Firstly, it binds Mn2+ at a site dissimilar to the presumed metal binding sites of Fur family proteins, and resolving this issue should give insight as to how it has adapted to be a Mn2+ sensor. Secondly, the demetallated form of Mur is active and controls a different regulon than the Mn2+-loaded protein. We will analyze this novel function. (3) Determine the molecular basis for the integration of iron and manganese metabolism. Preliminary evidence shows that B. japonicum senses and responds to a simultaneous deficiency in both iron and manganese at the level of transcription that is qualitatively different from individual deficiencies in each metal. We will focus initially on two operons involved in branched chain amino acid degradation to gain insight into this global response. PUBLIC HEALTH RELEVANCE: The ability of a bacterium to successfully infect and colonize its host requires it to adapt to that new environment, which includes the acquisition of nutrients that may be limiting. Bradyrhizobium japonicum is a model bacterium, but is taxonomically related to pathogens that are refractory to experimental analysis. We are studying novel mechanisms of manganese regulation and metabolic control in B. japonicum towards the end of understanding molecular strategies that bacteria employ for adaptation.